Aging is a complex process associated with gradual accumulation of macromolecular damage and loss of tissue regeneration. It has been difficult to tease out the relative contributions of these factors. p16Ink4a (p16) is a specific cell cycle inhibitor and tumor suppressor that accumulates with age and has been proposed as a biomarker of aging. p16 has been implicated in replicative senescence of many normal cell types in tissue culture, and we have shown that induction of p16 is sufficient to impose senescence in a cultured tumor cell line. Genetic analyses using p16-null mice revealed that p16 limits proliferation of progenitor cells in several tissues of older mice. However, whether p16 expression is sufficient to cause normal cells in vivo to arrest or senesce or to impose aging features is unknown. Surprisingly, none of p16's target Cyclin dependent kinases (Cdks) is essential in the mouse. To address these issues, we generated transgenic mice in which facile conditional expression of p16 is achieved by treatment with the antibiotic doxycycline (Dox). This simple system allows unprecedented control over cell proliferation. Induced p16 binds Cdk4 and inhibits proliferation of intestinal epithelial cells, including Lgr5+ intestinal stem cels. Broad induction in young mice imposes early aging phenotypes characterized by hair hypopigmentation and loss, weight loss, kyphosis, and anemia. We propose in Aim 1 to examine effects of selective p16 expression on aging parameters and cell proliferation in three different tissues--pancreatic islets, skin, and intestine. These tissues were selected for their ag-related phenotypes, evidence for roles for endogenous p16 in age-related dysfunction, established transactivator lines for Dox-sensitive transgene induction, and genetically validated histological markers of stem cells. In Aim 2, we will test whether the p16-imposed aging features and cell cycle arrest are reversible. These studies will establish that loss of cell proliferation s sufficient to confer aging phenotypes, challenging the paradigm that macromolecular damage is needed. Further, this work will explore the fundamental reversibility of such aging features while defining their relationship to stem/progenitor cell arrest and senescence. PUBLIC HEALTH RELEVANCE: This proposal examines in mouse models the hypothesis that the tumor suppressor and cell cycle inhibitor p16Ink4a is an effector of aging and age-related organ dysfunction. We propose to test whether p16-mediated cell cycle inhibition is sufficient to impose signature features of aging, whether defects in tissue stem cell proliferation are involved, and whether these features are fundamentally reversible.